PROJECT SUMMARY The discovery and repurposing of CRISPR-Cas enzymes for genome and transcriptome manipulation has profoundly impacted the pace, breadth, and depth of experimental design and investigation in the biological and medical sciences. The search for distinct types of CRISPR-Cas enzymes continues to uncover novel chemical mechanisms and biological roles that make these proteins well suited for new types of investigative and therapeutic applications. For example, several Cas9 orthologs were recently shown to bind and cleave RNA in an RNA-guided, protospacer adjacent motif (PAM)-independent manner, enabling in vivo repression of protein expression through targeted mRNA binding and even inhibiting RNA bacteriophage infection. Several Cas12a orthologs were recently found to act as non-specific single-stranded DNA (ssDNA) nucleases once activated by RNA-guided binding of target DNA, a property that could be used to interfere with life cycles of human pathogenic ssDNA parvoviruses or microbial ssDNA bacteriophage. Thus, these CRISPR-Cas enzymes continue to promise exciting, innovative RNA- and ssDNA-targeting applications beyond their established impactful implementation as genome editors. Before deploying these enzymes as medical tools, however, it is prudent to design and test mechanisms through which their nucleic acid-modifying and -binding activities can be rapidly activated or inactivated to prevent undesired editing. The objective of the proposed research is to develop allosterically regulated CRISPR-Cas enzymes to enable precise spatiotemporal control over next-generation genome and transcriptome modification. In the first aim, allosterically sensitive sites will be systematically mapped within a set of medically useful Cas12a and RNA-targeting Cas9 orthologs, yielding a panel of new CRISPR-Cas enzymes that are controlled by local administration of 4-hydroxytamoxifen (4-HT) or rapamycin. In the second aim, we will expand the chemical diversity of ligands and metabolites capable of exerting control over CRISPR- Cas enzyme activity by transplanting ligand-binding regulatory domains harvested from natural sensor proteins into allosterically sensitive sites in Cas9 and Cas12a orthologs. In the third aim, allosteric CRISPR-Cas molecular recorders will be deployed to quantify metabolic dysregulation across a heterogenous cell population, genetically encoding single cell metabolic profiles that are retrievable by deep sequencing. Success of these aims will set the stage for development of CRISPR-Cas enzymes that automatically sense and respond to dynamic profiles of defined chemical cue combinations, facilitating safe deployment of smart nucleic acid editors for therapeutic applications and for longitudinal reporting of intracellular ligand states using traditional reporters or through genome-encoded recording. UC Berkeley offers a collaborative, collegial, interdisciplinary, and scientifically rigorous environment that is conducive to highly effective postdoctoral scientific and professional training, and its established, world-class investigators possess the technical expertise to complement that provided in the Doudna lab and to ensure the success of the proposed fellowship training plan.